Doudna's confidence in CRISPR research potential burns hard

Are there any particular puzzles in bacteria that could lead you to a research tool?

It's always hard to predict. That said, I'll give you an example of an interesting phenomenon: the discovery of this new category of incredibly small bacteria. It is a brand new phylum of organisms – they are now called the phyla radiation candidate bacterium (CPR). They almost call into question the notion of cell and virus.

Many of these organisms probably grow in symbiosis with other insects, sharing important molecules, perhaps even the building blocks of DNA, RNA and proteins. But how important are molecules? How do they control their environment so that other types of bacteria do not grow and do not crowd them out?

These are all unanswered questions. We do not understand anything about their fundamental biology in the molecular sense. Will the answers to these questions lead to new technology? I do not know, but it will certainly lead to interesting biology.

So, the place to search for new research tools could be atypical organisms, so to speak?

But how do you define atypical? Here's Steve Forbert's old song: "It's often said that life is strange … but compared to what?

These small bacteria from CPR are those in which you and Jillian Banfield of the University of California at Berkeley recently discovered new enzymes Cas. [for cutting strands of DNA] that could be used with CRISPR technology, is not it? What makes these enzymes potentially potentially so interesting and useful?

One of the new enzymes we have identified is called "CasX". This is particularly interesting because it seems to work very differently from its cousin Cas9, the enzyme that many conventional bacteria use in their CRISPR defenses and which is commonly used in CRISPR technology. But some basic ingredients are the same. This gives us an overview of the basic recipe for CRISPR cutting proteins. The more we understand these proteins, the better we can design them. CasX is also attractive because it is much smaller than Cas9, which could facilitate insertion into cells for therapeutic editing of the genome.

CRISPR-Cas9 has also developed other derived technologies, such as CRISPR-GO, DNA imaging and anti-CRISPR. How could they help basic biology?

So let's go over these. CRISPR-GO is this clever way to use CRISPR enzymes to bring particular parts of the genome into physical proximity. There is evidence that when genes are expressed together in cells, they are often physically collected at the same place in the cells, which can fundamentally affect the protein levels produced by certain genes. CRISPR-GO provides technology for this type of physical connection, except that scientists can now control it rather than the cell controlling it. I think this creates an opportunity to begin to dissect the relationship between the 3D architecture of the genome and the communication between genes, and the resulting levels of proteins or RNA molecules that are made from these Genoa. So it's exciting. This is something that, once again, was not really possible before, to control the 3D architecture of chromosomes and to ask how this affects the output of the genome.

You mentioned DNA imaging. The idea is what is called "chromosome painting", in which you can program the CRISPR-Cas9 protein to bind and remain essentially sitting for long periods of time in certain parts of the DNA. You can decorate the CRISPR-Cas9 protein with different colors of dyes to illuminate a gene or section of a genome, even an entire chromosome, by simply tiling it with these small RNA-protein complexes. It is therefore an imaging method.

In the case of anti-CRISPRs, they are tiny natural proteins involved in the regulation of CRISPR systems. You can imagine that, in the case of virus-infected bacteria, the viruses have progressively evolved to prevent the CRISPR from eliminating them, notably by using these small inhibitors called anti-CRISPR. These are of interest because of the potential for controlling the results of gene editing – using this type of protein to turn off gene modification proteins in cells to protect the genome from unintentional changes. A great deal of research has been launched to examine natural regulators and inhibitors of CRISPR pathways and to question the possibility of exploiting them for technological purposes.

Could the development of anti-CRISPR alleviate fears about genome modification in humans or other organisms, if we had a switch to launch if CRISPR-Cas9 did not work as expected?

That's exactly what people think. In fact, there is a whole program funded by DARPA (Defense Advanced Research Projects Agency), entitled "Safe Genes," which discusses safe ways to manipulate genes and genomes. And one of the strategies that groups use for this is to use these anti-CRISPR.

Do you think CRISPR helps us better understand how all the parts of the cells work together rather than separately?

I think it will play more and more of that role in the future.

Let's go back to neuroscience because there is a case where CRISPR has become a priority in brain development studies. The researchers have not been able to determine the number of cell types present in the brain. We do not know how the brain develops in the direction of its 3D architecture. If you start with a stem cell or a few stem cells, how does it turn into a whole brain, and what is the map of the brain?

There is currently a lot of interest in using CRISPR to do what is called lineage mapping. If you have a cell population that is growing from a single cell or a small collection of cells, you can find out how the cells of this starting population are generating their offspring by introducing a little modification in their DNA to mark them.

Many research teams use CRISPR in this way to determine where these daughter cells end up in the brain and even what types of cells they become. I think this kind of experiments will lead to a more fundamental understanding of the development of tissues – especially the brain – that was not possible before.

This looks promising.

I will give you another example. There are interesting cases – and we find more and more cases when people get their DNA sequenced – from families in which everyone has a certain allele, a certain DNA sequence of a gene but only some of them have an associated disease. with this allele. Others do not do it. So you know that the DNA of unaffected people removes the negative impact of this gene and makes them less susceptible to cancer or any other disease that they would otherwise succumb to. What are these suppressors?

I think that understanding of this type of genetic interactions is going to be incredibly powerful in the future. Until now, we had not really found a way to do it because, first, people did not do many things to sequence their genomes. This is starting to happen more and more, with companies offering this and cost down. There is also a technology for the genetic manipulation of cells derived from the patient. So if you have someone who comes to a clinic and your disease is diagnosed, you can take that person's cells and grow them in the lab. This has been possible for a while, but before that, it was not possible to do genetics on these cells. We can now, in living cells that relate to a real patient.

This sounds like an unexpected benefit of sequencing technology.

I always like to point out that there is some serendipity in science. It's wonderful, but it also means you can not predict the results. CRISPR technology is an excellent example. If you had told me 10 years ago that bacteria had developed proteins that could be programmed to search for and cut any DNA sequence, I would have laughed well. I would have said, "Yes, it's really science fiction."

I think it's important for people to understand that this is how a lot of science is done.